The present invention relates to the treatment of melanoma, and in particular to treatment and diagnosis of anti-cancer therapy resistance in melanoma, as well as to the evaluation of resistance of a tumor or melanoma cells to anticer therapy.
The tendency of tumors to express resistance to therapeutic agents remains a major obstacle in cancer treatment. Resistance mechanisms can be classified as either physiological or cellular (see Bradley et al. (1988) Biochim. Biophys. Acta 948:87-128). Physiological resistance mechanisms refer to properties of the tumor such as vascularity, that limit drug penetration into the rumor. Cellular resistance mechanisms involve the ability of individual cancer cells to undergo mutations or other types of genetic alterations which biochemically render these cells more resistant to the cytotoxic effects of anti-cancer therapeutic drugs. Such mechanisms include decreased drug uptake, increased drug efflux and increased drug detoxification. Resistant cancer cells may arise en masse in a de novo fashion prior to drug treatment (xe2x80x9cintrinsic drug resistancexe2x80x9d) or they can be selected for by the drug (xe2x80x9cacquired drug resistancexe2x80x9d). One type of cancer which is well known for its intrinsic resistance to anti-ancer therapeutic drugs is malignant melanoma.
The incidence of malignant melanoma is increasing more rapidly than any other type of human cancer in North America (Armstrong et al. (1994) Cancer Surv. 19-20:219-240). Although melanoma is a curable cancer, the primary tumor must be removed at a very early stage of disease progression, i.e., before it has spread to distant sites. The presence of micrometastases can, and often do, lead to eventual symptomatic metastases. Because melanomas are intrinsically resistant to conventional methods of either chemotherapy or radiotherapy, it is virtually impossible to effectively treat such lesions in a clinically meaningful manner.
Intrinsic resistance to anti-cancer therapy, such as chemotherapeutic agents and radiotherapy, is a common characteristic of malignant melanoma and remains the main obstacle in clinical management of this disease. Thus, elucidating the molecular mechanisms underlying this intrinsic ability of melanoma tumors to resist anti-cancer therapy will lead to the development of clinically effective approaches to manage this disease.
Accordingly, there is considerable interest in elucidating the mechanisms by which cancer cells acquire resistance to anti-cancer therapies such as chemotherapy and radiation therapy. The rationale is that it ought to be possible to delay or prevent such resistance by pharmacologic means, provided more is known about the actual mechanisms involved. By way of example, it is known that some cancers may become resistant to commonly used drugs such as taxol, adriamycin, vincristine, and vinblastine by overexpressing a molecule known as P-glycoprotein on the cell surface. The function of P-glycoprotein is to pump potentially harmful drugs from the interior of the cell to the outside environment, to rid the cell of potentially harmful agents. Thus the pharmaceutical industry has put considerable resources into developing drugs which block the pumping action of P-glycoprotein in the hope of using such drugs as xe2x80x9cchemosensitizersxe2x80x9d. In other words, a cancer cell which overexpresses P-glycoprotein, and which therefore is resistant to taxol, for example, might be made drug sensitive by exposure of the cells to taxol plus an agent which blocks P-glycoprotein function. An example of a drug for which melanoma is resistant to treatment with cis-diamminedichloroplatinum(II) (CDDP), a DNA-alkylating anti-cancer therapeutic agent used in the treatment of many types of cancer.
Studies over the past decade have demonstrated that CDDP exerts cytotoxic effects by covalently binding to DNA, so as to induce DNA strand breaks (single or double-strand breaks), disturb local DNA structures, and form DNA intra- and inter-strand crosslinks, which interfere with DNA replication and mRNA transcription, and leads to apoptotic cell death. Thus, it has been postulated that genes conferring resistance to CDDP are involved in various aspects of DNA repair, anti-apoptotic pathways and/or drug detoxification. Indeed, in certain CDDP resistant cancer cells, decreased intracellular accumulation of CDDP (Bernal et al. (1990) Mol. Cell. Biochem. 95:61-67), up-regulation of glutathione or glutathione-transferase activity, increased levels of metallothioneins, enhanced DNA repair activity, and decreased sensitivity to apoptosis have been corelated with the acquisition of drug resistance. However, the significance of these pathways in the intrinsic resistance of melanomas have not been clearly demonstrated. Furthermore, increases in the expression of these factors were not always detected in CDDP-resistant tumor cells, suggesting that other mechanisms may be involved in the expression of CDDP resistance.
It is known that tyrosinase-related protein 2 (TYRP2) and other melanocytic proteins, such as tyrosinase and TYRP1, are involved in the enzymatic processes that convert L-tyrosine to the pigment melanin in skin melanocytes. TYRP2 is also known as L-DOPAchrome tautomerase (dct) and is referred to in the literature under other abbreviations, such as Trp-2 and Trp2. TYRP2 is a member of the tyrosinase gene family and, along with tyrosinase and TYRP1, function in the conversion of L-tyrosine to melanin. Specifically, TYRP2 catalyzes the conversion of L-DOPAchrome to 5,6-dihydroxyindole-2-carboxylic acid in the melanin biosynthetic pathway in melanocytes. In melanoma cells, increased expression of TYRP2 is well documented. Indeed, TYRP2 has been shown to be recognized by cytotoxic T lymphocytes as a melanoma associated tumor antigen and as such is currently being analyzed as a vaccine.
Exploitation of the mechanisms for anti-cancer therapy resistance promises to provide new treatment opportunities. There is a need for methodologies which harness and traverse the mechanisms of chemotherapy and radiotherapy resistance to render melanoma cells susceptible to conventional anti-cancer therapies, as well as to those therapies as yet undiscovered. Further, there is a need for evaluative methodologies in order to determine relative susceptibility of a melanoma to a therapy.
U.S. Pat. No. 5,831,016 discloses TYRP2 as a human tumor antigen recognized by cytotoxic T lymphocytes. While this document discusses the use of TYRP2 peptides as antigens, it does not teach or suggest treatments involving down-regulation of the expression of the TYRP2 gene, nor does the document relate to inactivation or reduction of TYRP2 protein products in melanoma cells.
It is an object of the present invention to provide a method for treatment of melanoma, particularly by reducing or otherwise traversing anti-cancer therapy resistance, which method obviates or mitigates one or more of the shortcomings of prior art methodologies.
Further objects of the invention are to provide a method for diagnosis of anti-cancer therapy resistance, and a method for evaluation of resistance of melanoma to candidate anti-cancer therapies.
In accordance with the invention, there is provided a method of treating melanoma in a patient comprising the steps of: a) down-regulating TYRP2 in melanoma cells; and b) administering an effective dose of an anti-cancer therapy to the patient.
The invention also provides a method of determining anti-cancer therapy resistance in melanoma cells comprising the steps of: a) obtaining melanoma cells; b) measuring TYRP2 in the melanoma cells; c) comparing TYRP2 measured in step b) to a pre-determined standard to obtain a measurement of anti-cancer therapy resistance in the melanoma cells.
In accordance with a further embodiment, the invention provides a method of evaluating an anti-cancer therapy for resistance in melanoma cells comprising the steps of: a) obtaining an initial population of melanoma cells; b) altering TYRP2 in a sub-set of the initial population of melanoma cells to form an altered population of melanoma cells; c) providing an anti-cancer therapy to the initial population and the altered population of melanoma cells; d) comparing efficacy of the anti-cancer therapy in the altered population of melanoma cells relative to the initial population of melanoma cells.
The invention addresses a need for reduced resistance to anti-cancer therapy regimes such as chemotherapy and radiotherapy in melanoma cells so as to render melanoma cells drug or radiation sensitive. In addition, the invention addresses a need for diagnostic methods capable of evaluating the relative sensitivity of a melanoma to an anti-cancer therapy, which will advantageously assist in determining the most appropriate course of treatment. Further, the invention provides a method by which candidate anti-cancer therapies may be evaluated for TYRP2-mediated resistance, which will advantageously assist in identifying therapies to which melanoma cells are less likely to illustrate resistance.